Until recently hereditary ovarian carcinoma has been attributed almost entirely to mutations in the BRCA1 and BRCA2 genes, with a much smaller contribution from mutations in DNA mismatch repair genes. However, there now is growing acceptance that rare mutations may substantially impact ovarian cancer risk, and account for a significant proportion of the ‘missing heritability’ of ovarian cancer.
While mutations in genes other than BRCA1 and BRCA2 are each individually rare, together they make up a significant proportion of cases. With at least 16 genes implicated in hereditary ovarian cancer to date, comprehensive testing for ovarian cancer risk is likely to utilise assessment of many genes. The falling cost of genomic sequencing and new advances in genomic technologies increase the feasibility of comprehensive evaluation of multiple genes simultaneously at low cost. Improved recognition of inherited risk will identify individuals who are candidates for targeted prevention.
Homologous recombination (HR) is a mechanism for repairing stalled replication forks, DNA interstrand crosslinks and double-strand breaks. Constitutional inactivating mutations in several genes that encode proteins crucial for DNA repair by HR have been shown to predispose to cancer. In particular, there is a strong association with female cancers and mutations in genes such as BRCA1, BRCA2, ATM, BRIP1, CHEK2, PALB2, RAD50 and RAD51C have been shown to confer susceptibility to breast and/or ovarian cancer. Indeed, the analysis of families with breast and ovarian cancer was crucial to the mapping of the BRCA1 gene. For many years, it was widely believed that the genetic contribution to families with breast and ovarian cancer was largely attributable to mutations in BRCA1 and BRCA2. However, RAD51C mutations have recently been identified in breast-ovarian cancer families. This suggested that analysis of such families may still have utility in cancer predisposition gene discovery.
In eukaryotic cells, DNA repair by HR involves several proteins of which a central player is the DNA recombinase RAD51, the ortholog of bacterial RecA. RAD51 forms helical filaments on DNA and catalyzes DNA strand invasion and exchange. Multiple other proteins are involved in these processes including five RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3.
In a first aspect the invention provides a method of determining that an individual is susceptible to cancer, the method comprising assaying a sample comprising a RAD51D-encoding nucleic acid molecule, or a complement thereof, from a human subject for the presence of an inactivating mutation in said nucleic acid, wherein the presence of an inactivating mutation in the nucleic acid indicates that the individual is susceptible to cancer.
In a second aspect, the invention provides a method of predicting a likelihood of a human subject developing cancer, the method comprising assaying a sample comprising a RAD51D-encoding nucleic acid molecule, or a complement thereof, from the human subject for the presence of an inactivating mutation in said nucleic acid, wherein the presence of an inactivating mutation in said nucleic acid indicates that the individual has an increased likelihood of developing cancer.
In a third aspect, the invention provides a method of treatment, the method comprising determining that a patient has cancer, assaying a sample comprising a RAD51D-encoding nucleic acid molecule, or a complement thereof, from the patient for the presence of a mutation in said nucleic acid, and, where a mutation is found in said nucleic acid, treating the patient using a DNA damaging agent or Topoisomerase I (TOPO I) inhibitor.
In a fourth aspect, the invention provides a method of treatment, the method comprising determining that a patient has cancer, assaying a sample comprising a RAD51D-encoding nucleic acid molecule, or a complement thereof, from the patient for the presence of an inactivating mutation in said nucleic acid, and, where an inactivating mutation is found in said nucleic acid, treating the patient using a poly (ADP-ribose) polymerase (PARP) inhibitor.
In a fifth aspect, the invention provides a method of genotyping a human subject, the method comprising assaying a sample comprising a RAD51D-encoding nucleic acid molecule, or a complement thereof, from a human subject for the presence of an inactivating mutation in said nucleic acid. In a suitable embodiment the inactivating mutation may be selected from the group consisting of: c.363delA; c.803G>A; c.480+1G>A; c.345G>C; c.556C>T; c.757C>T; c.270-271dupTA; and optionally c.748delC.
In a sixth aspect, the invention also provides a kit comprising oligonucleotides capable of amplifying an inactivating mutation in a RAD51D-encoding nucleic acid molecule, or a complement thereof, from a human subject. A kit in accordance with this fifth aspect of the invention may be of use in the various methods of the invention.
In a seventh aspect, the invention provides a system for determining a predisposition to cancer in a subject, comprising: (i) a sample analyzer for determining the RAD51D gene status in a sample from the subject, wherein the sample analyzer contains the sample, DNA extracted from the sample, RNA expressed from a RAD51D gene in the sample, complementary DNA synthesized from the RNA, DNA amplified from such extracted DNA and/or complementary DNA; (ii) a computer program for receiving the RAD51D gene status data for the sample; and (ii) a computer program for comparing the RAD51D gene status data for the sample to the reference RAD51D gene status associated with a predetermined degree of predisposition to cancer.
An eighth aspect of the invention is a diagnostic system, comprising: a polynucleotide sequence, the sequence including; a sample intake suitable for the intake of tissue, blood, or cells; a first reservoir in fluid contact the sample intake, the first reservoir includes a buffer and at least one pair of complementary polynucleotide primers that have at least 90 percent identity to at least two polynucleotides selected from the group consisting of SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, and SEQ ID NO.: 24; a second reservoir in fluid contact with the sample intake, the second reservoir includes a second buffer and a nucleic acid polymerase; a mixing chamber, in fluid contact with the sample intake, the first reservoir, the second reservoir; and the nucleic acid polymerase, where the mixing chamber mixes and incubates a mixture of the sample, the polynucleotide primers; and the nucleic acid polymerase, until the mixture amplifies a polynucleotide having 5′ and 3′ ends comprising said two polynucleotide primers to produce amplified polynucleotide; a mechanism for determining a sequence of nucleotide bases in the amplified polynucleotide, the mechanism is in fluid contact with the mixing chamber; a controller, the controller includes a compilation of the sequence of the amplified polynucleotide; a register of RAD51D (SEQ ID NO.: 1) truncation mutations, including at least one RAD51D truncation mutation selected from the group consisting of: c.363delA; c.803G>A; c.480+1G>A; c.345G>C; c.556C>T; c.757C>T; c.270-271dupTA; and c.748delC; a processor, the processor matches the sequence of the amplified polynucleotide to the truncation mutation in the register; and produces an output; the output includes: the sequence of the amplified polynucleotide; and a match between the sequence and any mutation in the register if the match exists.
SEQUENCE LISTINGSEQ ID NO: 1161121181241301361421481541601661721781841901961102110811141120112611321138114411501156116211681174118011861192119812041210121612221228124012401SEQ ID NO.: 2MGVLRVGLCPGLTEEMIQLLRSHRIKTVVDLVSADLEEVAQKCGLSYKALVALRRVLLAQ FSAFPVNGADLYEELKTSTAILSTGIGSLDKLLDAGLYTGEVTEIVGGPGSGKTQVCLCM AANVAHGLQQNVLYVDSNGGLTASRLLQLLQAKTQDEEEQAEALRRIQVVHAFDIFQMLD VLQELRGTVAQQVTGSSGTVKVVVVDSVTAVVSPLLGGQQREGLALMMQLARELKTLARD LGMAVVTNHITRDRDSGRLKPALGRSWSFVPSTRILLDTIEGAGASGGRRMACLAKSSR QPTGFQEMVDIGTWGTSEQSATLQGDQT; source Homo sapiens. SEQ ID NO.: 3 GCCTCCTCCTCTCTCCTTTC, 5′-3′, synthetic primer.SEQ ID NO.: 4 CACCCTTCCTGAGCCTCTC, 3′-5′, synthetic primer.SEQ ID NO.: 5 GGGTAGAATTGACACCCCATT, 5′-3′, synthetic primer.SEQ ID NO.: 6 TGACTTCTGACTCCAAGTGACC, 3′-5′, synthetic primer.SEQ ID NO.: 7 AAAGGGAGCAGAGGGTTCTC, 5′-3′, synthetic primer.SEQ ID NO.: 8 ATGTCCTGACCCCTTTCCTT, 3′-5′, synthetic primer.SEQ ID NO.: 9 TGGCCAGTGATGTTCAAAGA, 5′-3′, synthetic primer.SEQ ID NO.: 10 CCCATTAGTACGCTGAAGCTC, 3′-5′, synthetic primer.SEQ ID NO.: 11 GGACTCAGCCCATTTGTGTT, 5′-3′, synthetic primer.SEQ ID NO.: 12 AGCAAGTTTGAAGGCAAGGA, 3′-5′, synthetic primer.SEQ ID NO.: 13 CTGAGTCCTTGCATCCAGGT, 5′-3′, synthetic primer.SEQ ID NO.: 14 ATTGCACATCTGCATTTCCA, 3′-5′, synthetic primer.SEQ ID NO.: 15 CTTGCTGTATTTGGGATGGG, 5′-3′, synthetic primer.SEQ ID NO.: 16 TTTGGGGTTCAGAAGCTGAC, 3′-5′, synthetic primer.SEQ ID NO.: 17 CTCTCCGTAAAATGAAGCGG, 5′-3′, synthetic primer.SEQ ID NO.: 18 TAAACAGCAGGCGTTACTGG, 3-5′, synthetic primer.SEQ ID NO.: 19 CAGAACCAGTGCTTGAAAGAAA, 5′-3′, synthetic primer.SEQ ID NO.: 20 GGCCTCACATGTACCTGAGTT, 3′-5′, synthetic primer.SEQ ID NO.: 21 GAATCTGGGCAAGGTTTGGT, 5′-3′, synthetic primer.SEQ ID NO.: 22 TGGGTTTTAGCCTGAAGCAG, 3′-5′, synthetic primer.SEQ ID NO.: 23 AGGCCTCTGTTTTCCTCTCC, 5′-3′, synthetic primer.SEQ ID NO.: 24 CGATGGTGTCCAGGAGAATC, 3′-5′, synthetic primer.